城市污水中病原性大肠埃希氏菌毒力基因eaeA和rfbE的实时荧光定量PCR检测

摘要

针对病原性大肠埃希氏菌EHEC和EPEC的毒力基因eaeA和rfbE,选用特异性引物,建立了实时荧光定量PCR检测方法.利用从污水中分离出的目的核酸片段构建重组质粒,通过测序和BLAST比对分析,确定了PCR扩增的特异性.将重组质粒作为模板,分别测定标准曲线,在eaeA基因模板量8.77～8.77×105 copy,rfbE基因模板量4.98 ～4.98×105copy的范围内,Ct(循环阈值)与模板量的对数值具有良好的线性关系[ R2为0.997( eaeA)和1.000(rfbE)].结合膜吸附洗脱浓缩方法,对于实际水样,eaeA和rfbE基因的检测限分别为1.75×102和9.96×101copy/100 mL.利用该方法对城市污水处理厂进、出水进行检测的结果显示,污水二级处理工艺对这2种毒力基因的去除效果明显.%For the pathogenic bacteria Escherichia coli, a quantitative detection method targeting the virulence genes eaeA and rfbE was established by real-time PCR technology. A recombinant plasmid was constructed by cloning purified DNA fragment from positive water sample into vector. By BLAST analysis, the specificity of amplification was identified. Utilizing the recombinant plasmid as the targeted gene standard, a good linear correlation between the Ct values and the logarithm of input copy numbers was found while the eaeA gene ranged from 8.77 to 8. 77 × 105 copy ( R2 =0.997), and the rfbE gene ranged from 4.98 to 4.98 × 105 copy ( R2 = 1.000). Combined with the concentration procedure, the detection limit was 1. 75 × 102 copy per 100 mL water sample for eaeA, and 9. 96 × 101 copy/100 mL for rfbE. The results indicated that the municipal wastewater treatment process could effectively remove these virulence genes.